Thermostable variants of neutral proteases of Bacillus stearothermophilus and Bacillus thermoproteolyticus

Abstract

PCT No. PCT/NL95/00037 Sec. 371 Date Sep. 19, 1996 Sec. 102(e) Date Sep. 19, 1996 PCT Filed Jan. 26, 1995 PCT Pub. No. WO95/20663 PCT Pub. Date Aug. 3, 1995The present invention provides genes encoding extremely thermostable variants of neutral proteases, as well as, vectors and cells comprising these genes and proteases produced through these genes, vectors and/or cells. In particular variants of the neutral proteases of Bacillus stearothermophilus and Bacillus thermoproteolyticus (thermolysine) and sequences and cells coding therefor are provided.

What is claimed is: 
     
       1. A method for producing a neutral protease having higher thermostability than thermolysin comprising site directed mutagenesis of the DNA coding for the neutral protease of SEQ ID NO: 3 at the weak regions which correspond to residues 1-25, 59-72, 106, and 189 of SEQ ID NO: 3, wherein said site-directed mutagenesis provides replacement of three amino acid residues located at residues 59, 66, and 72 or 61, 66, and 72 of SEQ ID NO: 3 in the N-terminal domain of said protease by the corresponding residue in thermolysin. 
     
     
       2. A polypeptide having metallo-endopeptidase activity obtainable by a method according to claim 1.